Hydroxyurea differentially modulates activator and repressors of γ-globin gene in erythroblasts of responsive and non-responsive patients with sickle cell disease in correlation with Index of Hydroxyurea Responsiveness

Hydroxyurea (HU), the first of two drugs approved by the US Food and Drug Administration for treating patients with sickle cell disease (SCD), produces anti-sickling effect by re-activating fetal γ-globin gene to enhance production of fetal hemoglobin. However, approximately 30% of the patients do not respond to HU therapy. The molecular basis of non-responsiveness to HU is not clearly understood. To address this question, we examined HU-induced changes in the RNA and protein levels of transcription factors NF-Y, GATA-1, -2, BCL11A, TR4, MYB and NF-E4 that assemble the γ-globin promoter complex and regulate transcription of γ-globin gene. In erythroblasts cultured from peripheral blood CD34+ cells of patients with SCD, we found that HU-induced changes in the protein but not the RNA levels of activator GATA-2 and repressors GATA-1, BCL11A and TR4 correlated with HU-induced changes in fetal hemoglobin (HbF) levels in the peripheral blood of HU high and low responders. However, HU did not significantly induce changes in the protein or RNA levels of activators NF-Y and NF-E4. Based on HU-induced changes in the protein levels of GATA-2, -1 and BCL11A, we calculated an Index of Hydroxyurea Responsiveness (IndexHU-3). Compared to the HU-induced fold changes in the individual transcription factor protein levels, the numerical values of IndexHU-3 statistically correlated best with the HU-induced peripheral blood HbF levels of the patients. Thus, IndexHU-3 can serve as an appropriate indicator for inherent HU responsiveness of patients with SCD

NF-Y Recruits Both Transcription Activator and Repressor to Modulate Tissue- and Developmental Stage-Specific Expression of Human γ-Globin Gene

The human embryonic, fetal and adult β-like globin genes provide a paradigm for tissue- and developmental stage-specific gene regulation. The fetal γ-globin gene is expressed in fetal erythroid cells but is repressed in adult erythroid cells. The molecular mechanism underlying this transcriptional switch during erythroid development is not completely understood. Here, we used a combination of in vitro and in vivo assays to dissect the molecular assemblies of the active and the repressed proximal γ-globin promoter complexes in K562 human erythroleukemia cell line and primary human fetal and adult erythroid cells. We found that the proximal γ-globin promoter complex is assembled by a developmentally regulated, general transcription activator NF-Y bound strongly at the tandem CCAAT motifs near the TATA box. NF-Y recruits to neighboring DNA motifs the developmentally regulated, erythroid transcription activator GATA-2 and general repressor BCL11A, which in turn recruit erythroid repressor GATA-1 and general repressor COUP-TFII to form respectively the NF-Y/GATA-2 transcription activator hub and the BCL11A/COUP-TFII/GATA-1 transcription repressor hub. Both the activator and the repressor hubs are present in both the active and the repressed γ-globin promoter complexes in fetal and adult erythroid cells. Through changes in their levels and respective interactions with the co-activators and co-repressors during erythroid development, the activator and the repressor hubs modulate erythroid- and developmental stage-specific transcription of γ-globin gene.</p

IDO Immune Status after Chemoradiation May Predict Survival in Lung Cancer Patients

Host immunity influences the impact of radiotherapy (RT) in cancer, but mechanistic connections remain obscure. In this study, we investigated the relationship of indoleamine 2,3-dioxygenase (IDO) systemic activity on clinical outcomes in RT-treated non-small cell lung cancer (NSCLC). IDO-mediated production of kynurenine and the kynurenine:tryptophan ratio in patient blood serum were determined for stage III NSCLC patients at times before, during, and after RT administration and then correlated to overall survival (OS), progression-free survival, and disease progression rate in patients. We found the impact of RT on these serum IDO markers to be heterogeneous in patients. On average, kynurenine:tryptophan ratios were reduced during RT but restored after RT. Notably, both baseline levels of kynurenine:tryptophan and changes in the levels of kynurenine after RT were significantly associated with OS. When combined, favorable change and favorable baseline corresponded with very long-term OS (median OS was not reached after 57 months of median follow-up). Favorable change combined with unfavorable baseline still corresponded with a lack of distant metastases. Our results suggest that RT alters IDO-mediated immune status in NSCLC patients and that changes in this serum biomarker may be useful to predict outcomes and perhaps personalize RT dosage to improve survival.Significance: Radiotherapy appears to influence systemic IDO activity and to exert a significant impact on metastatic risk and overall survival, with possible implications for defining a biomarker to optimize radiation dose in patients to improve outcomes. Cancer Res; 78(3); 809-16. ©2017 AACR

Oral Pathobiont Activates Anti-Apoptotic Pathway, Promoting both Immune Suppression and Oncogenic Cell Proliferation.

Chronic periodontitis (CP) is a microbial dysbiotic disease linked to increased risk of oral squamous cell carcinomas (OSCCs). To address the underlying mechanisms, mouse and human cell infection models and human biopsy samples were employed. We show that the \u27keystone\u27 pathogen Porphyromonas gingivalis, disrupts immune surveillance by generating myeloid-derived dendritic suppressor cells (MDDSCs) from monocytes. MDDSCs inhibit CTLs and induce FOXP3 + 

A facilitated tracking and transcription mechanism of long-range enhancer function

In the human ε−globin gene locus, the HS2 enhancer in the Locus Control Region regulates transcription of the embryonic ε-globin gene located over 10 kb away. The mechanism of long-range HS2 enhancer function was not fully established. Here we show that the HS2 enhancer complex containing the enhancer DNA together with RNA polymerase II (pol II) and TBP tracks along the intervening DNA, synthesizing short, polyadenylated, intergenic RNAs to ultimately loop with the ε-globin promoter. Guided by this facilitated tracking and transcription mechanism, the HS2 enhancer delivers pol II and TBP to the cis-linked globin promoter to activate mRNA synthesis from the target gene. An insulator inserted in the intervening DNA between the enhancer and the promoter traps the enhancer DNA and the associated pol II and TBP at the insulator site, blocking mid-stream the facilitated tracking and transcription mechanism of the enhancer complex, thereby blocking long-range enhancer function

Genetic Variations in the Transforming Growth Factor-β1 Pathway May Improve Predictive Power for Overall Survival in Non-small Cell Lung Cancer

Purpose: Transforming growth factor-β1 (TGF-β1), a known immune suppressor, plays an important role in tumor progression and overall survival (OS) in many types of cancers. We hypothesized that genetic variations of single nucleotide polymorphisms (SNPs) in the TGF-β1 pathway can predict survival in patients with non-small cell lung cancer (NSCLC) after radiation therapy. Materials and Methods: Fourteen functional SNPs in the TGF-β1 pathway were measured in 166 patients with NSCLC enrolled in a multi-center clinical trial. Clinical factors, including age, gender, ethnicity, smoking status, stage group, histology, Karnofsky Performance Status, equivalent dose at 2 Gy fractions (EQD2), and the use of chemotherapy, were first tested under the univariate Cox's proportional hazards model. All significant clinical predictors were combined as a group of predictors named "Clinical." The significant SNPs under the Cox proportional hazards model were combined as a group of predictors named "SNP." The predictive powers of models using Clinical and Clinical + SNP were compared with the cross-validation concordance index (C-index) of random forest models. Results: Age, gender, stage group, smoking, histology, and EQD2 were identified as significant clinical predictors: Clinical. Among 14 SNPs, BMP2:rs235756 (HR = 0.63; 95% CI:0.42-0.93; p = 0.022), SMAD9:rs7333607 (HR = 2.79; 95% CI 1.22-6.41; p = 0.015), SMAD3:rs12102171 (HR = 0.68; 95% CI: 0.46-1.00; p = 0.050), and SMAD4: rs12456284 (HR = 0.63; 95% CI: 0.43-0.92; p = 0.016) were identified as powerful predictors of SNP. After adding SNP, the C-index of the model increased from 84.1 to 87.6% at 24 months and from 79.4 to 84.4% at 36 months. Conclusion: Genetic variations in the TGF-β1 pathway have the potential to improve the prediction accuracy for OS in patients with NSCLC

In Human Beta-Globin Gene Locus, ERV-9 LTR Retrotransposon Interacts with and Activates Beta- but Not Gamma-Globin Gene

Abstract Retrotransposons including endogenous retroviruses and their solitary long terminal repeats (LTRs) comprise over 40% of the human genome. Many of them are located in intergenic regions far from genes. Whether these intergenic retrotransposons serve beneficial host functions was not known. In the human b-globin gene locus, an ERV-9 LTR retrtransposon is located near the 5’ end of the locus control region (LCR) at 40-70 kb upstream of the human fetal g- and adult b-globin genes. To address the functional significance of the intergenic ERV-9 LTR, we generated transgenic (Tg) mice carrying a 100 Kb BAC clone spanning the entire human b-globin gene locus from the ERV-9 LTR to b-globin gene and showed that the LTR retrotransposon serves long-range, beneficial host function (Pi et al., PNAS 2010): The ERV-9 LTR containing multiple CCAAT and GATA motifs competitively recruits high concentration of NF-Y and GATA-2 present in low abundance in adult erythroid cells to assemble an LTR/RNA polymerase II complex. Deletion of the ERV-9 LTR by Cre-loxP mediated in situ recombination in the BAC Tg mice suppresses transcription of b-globin gene but activates transcription of g-globin gene. The results indicate that the ERV-9 LTR activates transcription of b-globin gene in erythroid cells during development. Therefore, LTR deletion drastically suppressed b-globin gene and re-activated g-globin gene through a competitive mechanism of globin gene switching. Alternatively, the primary effect of the ERV-9 LTR could be to suppress g-globin gene during development. Therefore, LTR deletion re-activated g-globin gene, which then suppressed transcription of b-globin gene. To differentiate between these two possibilities, we utilized Mx1-Cre mice to conditionally delete the ERV-9 LTR in the erythroid cells of the adult BAC transgene mice, in which g-globin gene was already silenced and b-globin gene fully activated. If the primary target of the ERV-9 LTR enhancer complex was g-globin gene, deletion of the ERV-9 LTR should not be able to activate the already silenced g-globin gene nor to suppress the fully active b-globin gene. However, the same effect on transcriptional suppression of b-globin gene and re-activation of g-globin gene was observed. These results indicate that the primary target of the ERV-9 LTR is the b-globin gene and not the g-globin gene. The molecular factors underlying the preferential interaction between the ERV-9 LTR and the b-globin gene are under investigation and will be presented Disclosures No relevant conflicts of interest to declare. </jats:sec

Non-Coding RNAs Transcribed from ERV-9 LTR Retrotransposons Regulate Erythropoiesis

Abstract Long noncoding RNAs (lncRNAs) regulate diverse cellular processes in development, differentiation and malignancy. In human cells, over 80% of the lncRNAs contain retrotransposon sequences transcribed from Alu, L1 and LTR retrotransposons, which comprise ~40% of the human genome. The functional significance of the retrotransposon lncRNAs is largely unknown. The human genome contains ~4000 copies of the ERV-9 LTR retrotransposon, which exhibits strong enhancer activity and initiates synthesis of ERV-9 lncRNAs in erythroid progenitor cells. Recently, we discovered that depletion of the ERV-9 lncRNAs in human erythroid progenitor cells cultured ex vivo from peripheral blood CD34+ cells inhibited ex vivo erythropoiesis. Whole genome RNA sequencing (RNA-seq) found that depletion of ERV-9 lncRNAs significantly suppressed transcription of 608 genes including ~50 key erythroid genes. We hypothesize that the ERV-9 lncRNAs together with the ERV-9 LTR act in cis to regulate transcription of these key erythroid genes and other genes to set up a transcriptional network that promotes erythropoiesis. In the human b-globin gene locus, we showed previously that the ERV-9 LTR retrotransposon performs a beneficial biological function: The ERV-9 LTR enhancer binds NF-Y and GATA-1 and -2 to assemble an LTR-pol II transcription complex, which transcribes long, noncoding RNAs (lncRNAs) from the LTR R-U5 regions through the downstream locus control region (LCR) and intergenic DNAs to reach and activate transcription of b-globin gene 70 kb away. In this tracking and transcription (T&amp;T) mechanism of long-range LTR enhancer function, the ERV-9 lncRNAs could be merely by-products of the tracking and transcribing process of the LTR complex without any functional significance. However, we found recently that depletion of the ERV-9 lncRNAs suppressed transcription of the entire human globin gene locus, diminished occupancies of NF-Y, GATA-1 and -2 and pol II at the ERV-9 LTR and reduced the looping frequency of the ERV-9 LTR with the globin gene locus in erythroid progenitor cells. Thus, the ERV-9 lncRNAs acted in cis to facilitate assembly of the LTR-pol II complex and modulate long-range LTR enhancer function in transcriptional activation of b-globin gene. Genome-wide, whether the ERV-9 lncRNAs transcribed from other gene loci perform similar biological function is under investigation. Disclosures No relevant conflicts of interest to declare. </jats:sec

Abstract 687: Critical role and mechanism of WASF3 in HER2/HER3 regulation of breast cancer metastasis

Abstract WASF3 is overexpressed in high-grade breast cancer and promotes invasion and metastasis but does not affect proliferation. The HER2/ERBB2/NEU gene is also frequently overexpressed in breast cancer and has been shown to promote invasion and metastasis in these tumors. Here we show that WASF3 in present in the HER2 immunocomplex and suppression of WASF3 function leads to suppression of invasion even in the presence of HER2 expression. Overexpression of both HER2 and WASF3 in non-metastatic MCF7 breast cancer cells promotes invasion and metastasis more significantly than either gene alone. HER2 forms homodimers as well as heterodimers with other HER family members and we now show that the ability of WASF3 to promote invasion is highly dependent on the HER2/HER3 heterodimer. The engagement of WASF3 with the HER2/HER3 complex facilitates its phospho-activation and transcriptional upregulation, which is facilitated by HER2/HER3 activation of JAK/STAT signaling. In breast cancer cells overexpressing HER2, therefore, WASF3 is specifically required to facilitate the invasion/metastasis response. Targeting WASF3, therefore, could be a potential therapeutic approach to suppress metastasis of HER2-overexpressing breast tumors. Citation Format: yong teng, Wenhu Pi, John Cowell. Critical role and mechanism of WASF3 in HER2/HER3 regulation of breast cancer metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 687.</jats:p

Augmentation of the anticancer activity of CYT997 in human prostate cancer by inhibiting Src activity

Abstract Background Abnormalities of tubulin polymerization and microtubule assembly are often seen in cancer, which make them very suitable targets for the development of therapeutic approach against rapidly dividing and aggressive cancer cells. CYT997 is a novel microtubule-disrupting agent with anticancer activity in multiple cancer types including prostate cancer. However, the molecular mechanisms of action of CYT997 in prostate cancer have not been well characterized. Methods Src knockdown cells were achieved by lentiviral-mediated interference. The drug effects on cell proliferation were measured by MTS. The drug effects on cell viability and death were determined by Cell Titer-Glo® Luminescent cell viability kit and flow cytometry with Zombie Aqua™ staining. The drug effects on apoptosis were assessed by Cell Death Detection Elisa kit and Western blot with a cleaved PARP antibody. The drug effects on cell invasion were examined by Matrigel-coated Boyden chambers. Oxidative stress was detected by DCFH-DA staining and electrochemical biosensor. Mouse models generated by subcutaneous or intracardiac injection were used to investigate the in vivo drug efficacy in tumor growth and metastasis. Results CYT997 effectively inhibited proliferation, survival, and invasion of prostate cancer cells via blocking multiple oncogenic signaling cascades but not the Src pathway. Inhibition of Src expression by small hairpin RNA or inactivation of Src by dasatinib increased the CYT997-induced cytotoxicity of in vitro. Moreover, the combination of dasatinib and CYT997 exhibited a superior inhibitory effect on tumor growth and metastasis compared with either of the drugs alone. Conclusion Our findings demonstrate that blockage of Src augments the anticancer effect of CYT997 on prostate cancer and suggest that co-treatment of dasatinib and CYT997 may represent an effective therapeutic regimen for limiting prostate cancer